WO2022097716A1

WO2022097716A1 - Method for producing reversibly immortalized cell

Info

Publication number: WO2022097716A1
Application number: PCT/JP2021/040757
Authority: WO
Inventors: 光雄押村; 寿晃田畑; 康宏香月; 愛海宇野
Original assignee: 株式会社Trans Chromosomics; 国立大学法人鳥取大学
Priority date: 2020-11-06
Filing date: 2021-11-05
Publication date: 2022-05-12
Also published as: CN116419968A; EP4242317A1; US20230407268A1; JPWO2022097716A1

Abstract

The present invention addresses the problem of providing: a method for producing a reversibly immortalized cell, whereby it becomes possible to proliferate a cell having an immortalizing gene introduced therein for a long period without damaging the chromosome in the cell and it also becomes possible to remove the immortalizing gene; and a method for producing a large amount of reversibly immortalized cells which can be cloned and have stabilized quality.　According to the present invention, a method for producing a reversibly immortalized cell is provided, the method comprising: a step for introducing a chromosomally unintegrated RNA virus vector which carries at least one immortalizing gene selected from the group consisting of Bmi-1 gene, TERT gene and SV40T gene into a mammalian cell to express the immortalizing gene in the cell; and a step for culturing the cell to proliferate the cell.

Description

Method for producing reversible immortalized cells

The present invention relates to a method for producing a reversible immortalized cell, and more particularly to a method for producing a reversible immortalized cell using a chromosomal non-integrating RNA viral vector carrying a predetermined immortalizing gene.

The main cell sources used for regenerative medicine and cell therapy are somatic stem cells such as mesenchymal stem cells (MSC) and pluripotent stem cells such as ES cells and iPS cells. Among stem cells, mesenchymal stem cells (MSCs) are cells that originally exist in the body, so there is little risk of rejection and tumorigenesis, and they are excellent in terms of safety, but because the number of cell divisions is limited. The challenge is to secure a sufficient number of cells to supply to the medical field.

Therefore, attempts are being made to introduce an immortalizing gene into cells and allow them to proliferate indefinitely. As immortalizing genes, telomerase reverse transcriptase (TERT) genes, genes that regulate the expression or activity of telomerase (for example, Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.) are known. Has been done. Vectors such as plasmid DNA, lentiviral vector, retroviral vector, and adenoviral vector are used for introduction of these immortalizing genes into cells (Patent Document 1, Patent Document 2, Patent Document 3, etc.). The virus vector is preferable in that the gene can be efficiently introduced by simply filtering the culture supernatant of the recombinant virus vector-producing cell and adding it to the target cell. Among the above vectors, the plasmid DNA and the adenovirus vector have a vector genome of DNA and are integrated into the host chromosome. In the lentiviral vector and the retroviral vector, the vector genome is RNA, but in the cell, the DNA is taken in the form of DNA by a reverse transcription enzyme and integrated into the chromosome of the host to express the loaded gene. As described above, the vector generally used for immortalization has a risk of damaging the gene of the host cell by incorporating foreign DNA into the chromosome of the host cell and, in some cases, forming a tumor, and thus the safety of regenerative medicine. Has become a big problem in. On the other hand, the Sendai virus vector (SeV vector) is a non-chromosomally integrated RNA virus vector that does not take the form of DNA. The SeV vector can be introduced into a wide range of cell types and easily expresses proteins, so that it is widely used for inducing iPS cells (Patent Document 4). However, in the case of cells such as mesenchymal stem cells (MSC), which have a limited number of divisions and stop dividing after 2 months or more, it was recognized that long-term expression of the introduced gene is difficult with the SeV vector. The use of the SeV vector was limited to temporary high expression and was not used for cell transfer of immortalizing genes.

On the other hand, reversible (conditional) immortalized cells, in which an immortalizing gene can be introduced to immortalize a cell, and the immortalizing gene can be excised using a site-specific recombination enzyme after proliferation, have also been studied ( Non-Patent Document 1 etc.). However, even if the immortalizing gene is excised using a site-specific recombination enzyme, the obtained cells are genetically engineered cells, and there are concerns about adverse effects such as the formation of tumors in the host by such cells. However, it cannot be said that safety is ensured.

In addition, stem cells obtained from human tissues are a group of young cells and aged cells, and have various properties. They divide and age, so cells can be maintained at a constant quality. not. Therefore, there is a different problem that it is difficult to set quality standards, which are necessary elements for automation and mechanization.

Attempts to isolate only cells with the desired properties from those different cell populations are beginning to be discussed. Known methods for evaluating the quality of iPS cells include miRNA analysis, surface marker detection, cell morphology image analysis, and epigenome analysis, and MSC has also proposed the use of these techniques, but unlike iPS cells. Since MSCs do not grow indefinitely and cannot be cloned, they have not been put into practical use.

In addition, when separating MSCs with shallow passages without repeated division, a large amount of tissue containing MSCs is required, it is difficult to secure the required number of cells, which is a factor that raises the manufacturing cost. .. Therefore, treatment that requires a large number of cells is difficult, and treatment with autologous cells is the main treatment, and it is difficult to carry out inexpensive regenerative medicine using allogeneic cells.

Japanese Patent No. 3953399 WO 2017/0781 No. 176 Japanese Unexamined Patent Publication No. 2017-221219 Japanese Patent No. 5763340

INDUSTRIAL APPLICABILITY The present invention is a method for producing a reversible immortalized cell, which can proliferate the cell for a long period of time without damaging the chromosome of the cell into which the immortalizing gene has been introduced, and can remove the immortalizing gene. It is an object of the present invention to provide a method for obtaining a large amount of reversible immortalized cells that can be cloned and have stable quality.

As a result of diligent research to solve the above problems, the present inventors have obtained one or more immortalizing genes selected from the group consisting of Bmi-1 gene, TERT gene, and SV40T gene. When introduced into mesenchymal stem cells (MSC) by mounting it on the Sendai virus vector, which is a non-chromosomal RNA vector that has not been used for cell immortalization, surprisingly, it has been used for more than 80 days. It was found that cell proliferation does not occur and that infinite cell proliferation (immortalization) is possible, and that the rate of cell proliferation increases at an early stage after gene transfer. It was also confirmed that the produced immortalized cells had no chromosomal abnormality, had pluripotency, and could easily remove the immortalized gene by temperature control. Furthermore, the immortalized cells were successfully cloned from the obtained immortalized cell population, and 8 out of 10 clones of the cloned immortalized cells also showed normal nuclear type and were pluripotent. It was confirmed that immortalization can be induced in cells at any stage from young cells to senescent cells in which division has stopped, and that cloning is possible immediately after immortalization. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
[1] A method for producing a reversible immortalized cell, which comprises the following steps.
(1) A step of introducing a chromosome-non-integrating RNA viral vector carrying an immortalizing gene into mammalian cells and expressing the immortalizing gene in the cells, and (2) culturing the cells obtained in step (1). And the process of growing
[2] The method according to [1], wherein the immortalizing gene is one or more immortalizing genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.
[3] The method according to [1] or [2], wherein the immortalizing gene is any of the following (a) to (d).
(a) Combination of Bmi-1 gene, TERT gene, and SV40T gene
(b) Combination of Bmi-1 gene and TERT gene
(c) Combination of TERT gene and SV40T gene
(d) TERT gene
[4] The method according to any one of [1] to [3], wherein the cell is a somatic cell.
[5] The method according to [4], wherein the somatic cell is a somatic stem cell.
[6] The method according to [5], wherein the somatic stem cell is a mesenchymal stem cell.
[7] The method according to any one of [1] to [6], wherein the chromosomal non-integrated RNA viral vector is a negative strand RNA viral vector.
[8] The method according to [7], wherein the negative strand RNA virus vector is a paramyxovirus vector.
[9] The method according to [8], wherein the paramyxovirus vector is a Sendai virus vector.
[10] The method according to [9], wherein the Sendai virus vector is a temperature sensitive Sendai virus vector.
[11] The method according to [1], wherein the chromosomal non-integrating RNA virus vector is a Sendai virus vector, further comprising a step of removing the Sendai virus vector after culturing in the step (2).
[12] The method according to [11], wherein the removal of the Sendai virus vector is performed by changing the culture temperature from 35 ° C to 37 ° C.
[13] The method according to any one of [1] to [12], further comprising a step of cloning immortalized cells after culturing in the step (2).
[14] Immortalized cells obtained by the method according to any one of [1] to [13].
[15] Immortalized cells comprising a Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of Bmi-1 gene, TERT gene, and SV40T gene in a removable state.
[16] A regenerative medicine product comprising the immortalized cell according to [14] or [15].
[17] A temperature-sensitive Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.
[18] A kit for producing reversible immortalized cells, which comprises the temperature-sensitive Sendai virus vector according to [17].
This application claims the priority of Japanese Patent Application No. 2020-186146 filed on November 6, 2020, and includes the contents described in the specification of the patent application.

According to the present invention, by introducing an immortalizing gene into a cell using a non-chromosomal integrated RNA vector, a cell that is difficult to mass-culture, for example, a mesenchymal stem cell (MSC) used for regenerative medicine, is introduced into the chromosome. It is possible to immortalize (infinitely multiply) without damaging. This immortalization is possible regardless of the number of cell divisions to be immortalized and the number of cell divisions during immortalization. Further, by using a temperature-sensitive Sendai virus vector (SeV vector) as a non-chromosomal integrated RNA vector, the vector can be easily removed from cells only by changing the culture temperature without performing a complicated removal operation. Therefore, it is possible to supply a large amount of cells having excellent safety. In addition, the immortalized cells obtained by the present invention can be used for regenerative medicine because they have no chromosomal abnormality and have pluripotency as before gene transfer. In addition, it is difficult to clone non-immortalized cells because they have different lifespans and properties, but the immortalized cells produced by the present invention are easy to clone and can stably obtain high-quality cells. Therefore, the mechanization of cell manufacturing facilitates quality control, makes it possible to significantly reduce the manufacturing cost, which is an issue for industrialization, and contributes to the development of cell medicine and regenerative medicine.

The genomic structure of the SeV vector carrying the immortalizing factor gene is shown [A. SeV (18+) Bmi1 (HNL) OFP / TS15dΔF, B. SeV (PM) hTERT (HNL) EGFP / TS15ΔF, C. SeV (18+). ) SV40T / TS15ΔF, D. SeV (HNL) E6-E7, BFP / TS15ΔF]. Transmitted light and fluorescence (OFP) images of Bmi-1 solitary infected cells (MOI: 1,5,20), transmitted light and fluorescence (GFP) images of hTERT solitary infected cells (MOI: 1,5,20) show. The transmitted light image and the fluorescence (OFP, GFP) image of the two-factor (Bmi-1 / hTERT) co-infected cell (MOI: 1,5,20) are shown. The transmitted light image and the fluorescence (OFP, GFP) image of the three-factor (Bmi-1 / hTERT / SV40T) co-infected cell (MOI: 1,5,20) are shown. The cell morphology and cell condition determination 59 days after the introduction of immortalizing factors (No. 1 to No. 16) are shown. Shows the cell proliferation curve of immortalizing factors (No. 1, No. 2, No. 4, No. 12, No. 16) introduced cells (up arrow: morphological (transmitted light) image and fluorescent image point (Fig. 5)). , *: Telomere analysis points (FIGS. 11A, B), ▽: Chromosome analysis points). Shows cell proliferation curve of 3 factor (Bmi-1 / hTERT / SV40T) -introduced cells (NC: immortalized gene non-introduced, up arrow: morphology (transmitted light) image and fluorescence image point (Fig. 8), double arrow: morphology (Transmitted light) Image point (FIG. 9), *: Telomerase analysis point (FIG. 11B), ▽: Chromosome analysis point (FIG. 10). The transmitted light image and the fluorescence (OFP, GFP) image of the three-factor (Bmi-1 / hTERT / SV40T) -introduced cell are shown. The morphology (transmitted light) image of the three-factor (Bmi-1 / hTERT / SV40T) -introduced cell is shown (maintained at 35 ° C, temperature changed from 35 ° C to 37 ° C). The chromosomal analysis results (normal karyotype) after 90 days of subculture of the three-factor (Bmi-1 / hTERT / SV40T) -introduced cells are shown (chromosome analysis point (▽) in FIG. 7). The results of quantitative analysis of telomeres in long-term culture samples of immortalizing factors (No.1, No.2, No.4, No.12, No.16) are shown (A. Effects of immortalizing factor combinations on telomeres). (Day0, Day40: Telomere analysis point in Fig. 6 (*)), B. After introduction of factor 3 (Bmi-1 / hTERT / SV40T), change in telomere amount due to factor non-removal / removal (Day0, Day40: Fig. 6) Telomere analysis points (*), Day85, Day98: Telomere analysis points in Fig. 7 (*)). Pictures of adipocytes differentiated from early cultured MSCs and immortalized MSCs are shown. Pictures of osteoblasts differentiated from early cultured MSCs and immortalized MSCs are shown. Pictures of neurons differentiated from early cultured MSCs and immortalized MSCs are shown. Pictures of chondrocytes differentiated from early cultured MSCs and immortalized MSCs are shown. The cloning test result 1 of the immortalized cell population (2 weeks after the immortalization gene transfer) is shown (number in well: number of clones formed). The cloning test result 2 of the immortalized cell population (4 months after the immortalization gene transfer) is shown (number in well: number of clones formed). The cell proliferation curve of cloned immortalized cells (clone A10) is shown (▽: chromosome analysis point). The results of chromosomal analysis of cloned immortalized cells (clone A10) are shown (Day 80: Chromosome analysis point (▽) in FIG. 18). It shows the differentiation potential of cloned immortalized cells (fat cell differentiation, neuronal cell differentiation, osteoblast differentiation).

Cryopreservation points of MSCs used in single-cell cloning studies (early MSCs: Day9, mid-term MSCs: Day24, late MSCs: Day49) and immortalization induction points by SeV vector infection (early MSCs: Day21, mid-term MSCs: Day36, late MSCs) : Day61) is shown. Photographs of SeV vector infected cells (early MSCs, middle MSCs, late MSCs) and SeV vector non-infected MSCs (controls) before single-cell cloning (immediately before 96-well plate seeding) are shown. The results of a single cell cloning test of cell populations of SeV vector non-infected MSC (control) and SeV vector infected cells (early MSC) are shown (numbers in wells: number of clones formed, culture days: 2 weeks). The results of a single cell cloning test of a cell population of SeV vector-infected cells (medium-stage MSC, late-stage MSC) are shown (numbers in wells: number of clones formed, number of culture days: 2 weeks). Photographs of SeV vector infection test results for cells in which cell division has stopped (Day 90) (cells immediately before SeV vector infection, SeV vector non-infected cells (control cells), and SeV vector infected cells (2 weeks after infection)) ). The test results of single-cell cloning of arrested cells (Day 90) that started reproliferation by SeV vector infection are shown (numbers in wells: number of clones formed, number of culture days: 2 weeks). The cell proliferation curve of adipose tissue-derived human MSC (non-infected cell line, SeV vector infected cell line) in serum-free culture is shown. The cell proliferation curve of bone marrow-derived human MSC (non-infected cell line, SeV vector infected cell line) in serum-free culture is shown. A transmitted light image and a fluorescence (OFP, GFP) image of a SeV vector-infected cell line of a serum-free cultured adipose tissue-derived human MSC (hMSC-AT) and a SeV vector-infected cell line of a bone marrow-derived human MSC (hMSC-BM) are shown. (HMSC-AT: 16th and 58th days from the date of infection, hMSC-BM: 24th and 50th days from the date of infection). The cell proliferation curve of rat MSC (non-infected cell line, SeV vector infected cell line # 1, # 2) is shown. Transmission light images and fluorescence (OFP, GFP) images of SeV vector-infected cell lines of immortalized rat MSC are shown (2nd, 9th, and 58th days from the day of infection). The cell proliferation curve of HFL1 (non-infected cell line # 1, # 2, SeV vector infected cell line # 1, # 2, 37 ° C cultured SeV vector infected cell line # 1, # 2) is shown. Shown are transmitted light images and fluorescence (OFP, GFP) images of immortalized HFL1 [35 ° C culture (10 days, 34, 255 days from infection date), 35 ° C culture + 37 ° C culture (34 days + 221 days from infection date)]. .. The cell proliferation curve of HUVEC (non-infected cell line # 1, SeV vector infected cell line # 1, # 2 cultured at 35 ° C, SeV vector infected cell line # 1, # 2 cultured at 37 ° C) is shown. Immortalized HUVEC [35 ° C culture (7 days, 36, 72 days from infection date), 35 ° C culture + 37 ° C culture (35 days + 37 days from infection date)] transmitted light images and fluorescence (OFP, GFP) images are shown. .. It is a photograph showing the result of FISH analysis of immortalized MSC by introduction of a multiproliferative factor-inserted human artificial chromosome vector (in the lower right frame: DAPI-stained image of arrow cells). The results of a healing effect test of an enteritis model by transplantation of immortalized MSCs are shown.

1. 1. Method for Producing Reversible Immortalized Cells The present invention is a method for producing reversible immortalized cells, wherein the method (1) introduces a chromosomal non-integrating RNA virus vector carrying an immortalizing gene into mammalian cells. Then, the step of expressing the immortalizing gene in the cell and the step of (2) culturing and proliferating the cell obtained in the step (1) are included.

(cell)
In the present invention, the "cell" refers to all somatic cells other than germline cells (egg and sperm, oocyte, ES cell, etc.) and totipotent cells (iPS cells). Further, the "somatic cell" may be any of a primary cultured cell, a subcultured cell, and an established cell line. Further, the somatic cells may be naturally derived or artificially produced by differentiating from iPS cells or the like. Specific examples of somatic cells include cells that form tissues (fat cells, fibroblasts, nerve cells, skin cells, blood cells, muscle cells, osteoblasts, cartilage cells, hepatocytes, pancreatic cells, and renal cells. , Myocardial cells, brain cells, lung cells, splenocytes, adrenal cells, gingival cells, root membrane cells) or their precursor cells, immune system cells (B cells, T cells, monocytic cells) (Cells, etc.), somatic stem cells [mesophageal stem cells (adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord blood-derived stem cells, placenta-derived stem cells, etc.), hematopoietic stem cells, nerve stem cells, epidermal stem cells, intestinal epithelial stem cells, dental pulp stem cells, tooth roots, etc. Membrane stem cells, etc.] and the like. The origin of the cells is not particularly limited as long as it is a mammal, and examples thereof include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses.

(Reversible immortalized cells)
The "immortalized cell" refers to a cell whose proliferation does not stop even if cell division is repeated, that is, a cell having an infinite proliferation ability, unlike a primary cultured cell or a cell cultured under normal culture conditions. The "immortalized cell" in the present invention means a cell capable of infinite proliferation by introducing a predetermined immortalizing gene, and a cell whose infinite proliferation ability does not decrease even if it is repeatedly subcultured and cultured. .. The "immortalized cells" according to the present invention have different growth rates and growth periods depending on the origin of the cells or the culture conditions, but as a result of subculture, untreated cells proliferate or cease under the same culture conditions. It is possible to continue exponential growth for 20 days or more, preferably 60 days or more, more preferably 80 days or more even after the period. In addition, the immortalized cells of the present invention include both the cell population capable of infinite proliferation as described above and the immortalized cell line cloned from the cell population. In addition, "reversible immortalization" means that a cell proliferation is stopped or attenuated by introducing an immortalizing gene into a cell, making the cell proliferative indefinitely, and then removing the immortalizing gene. It means to let.

"Immortalization" means to release the limit of the number of cell divisions and cell aging of early cells, and to impart continuous cell division ability and proliferation ability. Specifically, under standard cell culture conditions, usually 5 or more passages, preferably 7 or more passages, 8 or more passages, 9 or more passages, 10 or more passages, 12 or more passages, 15 passages or more. As mentioned above, it means that more than 20 passages have become possible. Cells having a confluent number of passages of 0 can be amplified and cultured by subculturing using a method known to those skilled in the art. The cells obtained by one passage operation are called "passage number 1 (or second generation)" cells, and "

passage number

2, 3, 4 ..." corresponding to the number of passage operations. It can be expressed as "n (n (integer) is the number of passages) (n + 1th generation)". In addition, a step of freezing the cells may be included between each passage operation.

(Immortalization gene)
The immortalized cell of the present invention is produced by introducing a predetermined immortalizing gene into a cell using a chromosome non-integrating RNA viral vector. Here, the "immortalizing gene" refers to a gene that immortalizes a cell to acquire infinite proliferation ability and does not induce cell death. The immortalizing gene is an extrinsic gene and means an immortalizing gene newly introduced from the outside of the cell. Further, the immortalizing gene may be an immortalizing gene derived from other than human, or may be an immortalizing gene modified into a form expressible in a target cell. In the present invention, as the immortalizing gene, one or more genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene can be used, but a combination of the two genes can be used. Preferably, a combination of three genes is more preferred. When one kind of gene is used, the TERT gene is preferable, and examples of a preferable combination when two or more kinds of genes are combined include a combination of Bmi-1 gene and TERT gene, and a combination of TERT gene and SV40T gene.

In the present invention, unless otherwise specified, the "gene" includes not only a structural gene that defines the primary structure of a protein but also a region on a nucleic acid having a control function such as a promoter or an operator. Therefore, in the present invention, the term "gene" is used without distinguishing between a regulatory region, a coding region, an exon, and an intron, unless otherwise specified.

The "Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog) gene" has two nuclear localization signals and is a polycomb repressive complex 1: PRC1 localized in the cytoplasm or nucleus. It is a gene that encodes a protein that functions as one. As one of PRC1, Bmi-1 is involved in the regulation of expression of various genes including Hox gene by controlling chromatin remodeling and histone modification. Bmi-1 has the effect of controlling cell proliferation by suppressing the expression of p16 and p19Arf involved in the cell cycle, and plays an important role in maintaining self-renewal by being involved in cell division of hematopoietic stem cells and nerve stem cells. It is known to play.

"TERT (telomerase reverse transcriptase) gene" is a gene encoding telomerase reverse transcriptase (TERT). Telomerase RNA component (TERT) extends specific repeat sequences of eukaryotic chromosome ends (telomeres) from telomere RNA components (Telomerase RNA: TR or Telomerase RNA component: TERC) and other regulatory subunits. It constitutes the enzyme telomerase. It is known that TERT has a role in maintaining telomere length, whereas cell aging has a telomere length monitoring mechanism and cell senescence is caused by telomere shortening.

The "SV40T (simianvirus 40large Tantigen) gene" is a gene encoding the Simian virus 40 large T antigen. The genome of SV40 (simianvacuolating virus40) is divided into an early region that is expressed immediately after infection, a late region that is expressed during replication of the viral genome after infection, and a regulatory region that includes transcriptional regulation and the origin of replication. The early region encodes a large T antigen involved in the initiation of viral genome replication and inactivation of p53 and pRB, which are tumor suppressor gene products, and a small t antigen that binds to and inhibits the protein dephosphorylation enzyme PP2A.

A specific example of the Bmi-1 gene used in the present invention is the mouse BMI1 gene (SEQ ID NO: 1), a specific example of the TERT gene is the human TERT gene (SEQ ID NO: 2), and a specific example of the SV40T gene is SV40 large. The T antigen gene (SEQ ID NO: 3) can be mentioned. The Bmi-1 gene, TERT gene, and SV40T gene may also be these transcriptional variants, splicing variants and their orthologs.

The above Bmi-1 gene, TERT gene, and SV40T gene are 80% or more, preferably 90% or more, relative to each of the nucleotide sequences of SEQ ID NOs: 1, 2, and 3 as long as they have the same functions and activities. , More preferably a gene consisting of a base sequence having 95% or more sequence identity, or several (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 20) in each of the base sequences of SEQ ID NOs: 1, 2, and 3. May be a gene in which 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3) bases are substituted, inserted, added and / or deleted, and such homolog genes may also be used. It shall be included in the immortalizing gene referred to in the present invention. In addition, the Bmi-1 gene, TERT gene, and SV40T gene are artificially modified so that the product of the gene is expressed as a fusion protein with other proteins or peptides as long as it has the same function and activity. It may be a gene.

(Chromosome non-integrated RNA viral vector)
In the present invention, a "chromosome non-integrating RNA virus vector" is used as a vector for introducing and expressing the immortalization gene in cells. In the present invention, the viral vector means a vector having a genomic nucleic acid derived from the virus and capable of expressing the gene by incorporating a transgene into the nucleic acid. The "chromosome non-integrated RNA virus vector" is a virus vector derived from a virus and capable of introducing a gene into a target cell, and the introduced gene is integrated into a host chromosome (nuclear-derived chromosome). A non-hazardous carrier.

Examples of the chromosomal non-integrating RNA viral vector used in the present invention include a negative strand RNA viral vector. "Minus strand RNA virus vector" refers to a vector consisting of a virus containing RNA of minus strand (antisense strand complementary to the sense strand encoding the viral protein) as a genome, and minus strand RNA is also called negative strand RNA. .. As the negative-strand RNA virus used in the present invention, a single-stranded negative-strand RNA virus (also referred to as a non-segmented negative-strand RNA virus) is particularly preferable. "Single-stranded negative-strand RNA virus" refers to a virus having a single-stranded negative-strand (minus-strand) RNA in its genome, for example, Paramyxoviridae; Paramyxovirus, Morbillivirus, Rubulavirus, Pneumovirus, etc. Includes), Rhabdoviridae (including Rhabdoviridae; Vesculovirus, Lyssavirus, and Ephemerovirus genus), and viruses belonging to the family Filoviridae.

Examples of minus-strand RNA viruses that can be used in the present invention include Paramyxoviridae virus Sendai virus, Newcastle disease virus, Mumps virus, and measles virus. (Measles virus), RS virus (Respiratory syncytial virus), cattle epidemic virus (Rinderpest virus), distemper virus (Distemper virus), salparainfluenza virus (SV5), human

parainfluenza virus type

1,2,3, orthomixovirus family (Orthomyxoviridae) influenza virus (Influenza virus), Rhabdoviridae (Rhabdoviridae) vesicular stomatitis virus (Vesicular stomatitis virus), mad dog disease virus (Rabies virus) and the like can be mentioned, but Sendai virus (Sendai virus) is preferable.

(Sendai virus vector: SeV vector)
In a preferred embodiment of the present invention, a Sendai virus (SeV) vector is used as the non-chromosomal integrated RNA vector. The immortalizing factor genes of the Bmi-1 gene, TERT gene, and SV40T gene may be inserted separately into the SeV vector, or may be inserted together into a single SeV vector.

Sendai virus is a type of virus belonging to the genus Respirovirus of the Paramyxoviridae family, and contains a single minus strand (antisense strand against the sense strand encoding the viral protein) RNA as a genome.

The SeV vector is (i) highly efficient in gene transfer and expression into various mammalian cells including humans, and (ii) is a non-chromosomal non-integrating viral vector, and the vector is expressed in the cytoplasm. The introduced gene is not integrated into the host's chromosome, there is no risk of structural changes in the chromosome, (iii) it is not a human pathogenic virus, and (iv) the gene expression level is changed by changing the insertion position into the vector. It has features such as the ability to regulate and co-express multiple genes, and (v) the ability to remove the vector from the introduced cells after achieving the objectives.

The Sendai virus genome consists of NP (nucleocapsid) gene, P (phospho) gene, M (matrix) gene, F (fusion) gene, and HN (hemogglutinin / neuraminidase) in order from the 3'end to the 5'end. ) Gene and L (large) gene are included. Of these, Sendai virus can sufficiently function as a vector if it has the NP gene, P gene, and L gene, and can replicate the genome in cells and express the loaded gene. Since the Sendai virus has minus-strand RNA in its genome, the 3'side of the genome is upstream and the 5'side is downstream, which is the opposite of the normal case.

In the present invention, the SeV vector can be used as a natural strain, a wild strain, a mutant strain, or a commercially available product. The virus may be a virus having a structure similar to that of a virus isolated from nature, or a virus artificially modified by genetic recombination, as long as the desired function can be achieved. For example, any gene possessed by the wild-type virus may be mutated or deleted. Specifically, a non-propagating vector (ΔF) in which the F gene is deleted from the genome and no infectious particles are formed from the gene-introduced cells, and the F gene is deleted, and the M and / or HN gene is deleted. A vector that further deletes or has a mutation in the M and / or HN gene (eg, a temperature sensitive mutation) is preferably used in the present invention. Further, for example, a vector in which the F gene is deleted, the M or HN gene is further deleted, and the remaining M and / or HN gene is further mutated (for example, a temperature-sensitive mutation) is also preferably used in the present invention (patented). See No. 5763340, etc.).

Further, the SeV vector used in the method of the present invention is preferably temperature sensitive. "Temperature sensitivity" means that the activity is significantly reduced at a normal cell culture temperature (for example, 37 to 38 ° C) as compared with a low temperature (for example, 30 to 36 ° C). For example, Sendai virus TS7 (L protein Y942H / L1361C / L1558I mutation), TS12 (P protein D433A / R434A / K437A mutation, TS13 (P protein D433A / R434A / K437A mutation and L protein L1558I mutation), TS14 (P). Mutations such as protein D433A / R434A / K437A mutations and L protein L1361C) and TS15 (P protein D433A / R434A / K437A mutations and L protein L1361C / L1558I) are temperature-sensitive mutations and are preferred in the present invention. These mutations can be utilized. It is preferable to further introduce these mutations into the above-mentioned F gene-deficient SeV vector. For these SeV vectors, refer to Japanese Patent No. 5763340, WO2015 / 046229, etc. be able to.

The SeV vector in the present invention is a complex consisting of an infectious virus particle, a virus core, a complex of a virus genome and a virus protein, a non-infectious virus particle, and the like, and is loaded by introduction into cells. Includes a complex capable of expressing a gene that does. For example, a ribonucleoprotein (core part of a virus) consisting of a Sendai virus genome and Sendai virus proteins (NP, P, and L proteins) that bind to it expresses the introduced gene in the cell by introducing it into the cell. Can be done. The introduction into cells may be carried out by appropriately using a transfection reagent or the like. Therefore, such a ribonucleoprotein (RNP) is also included in the SeV vector in the present invention.

(Construction of SeV vector carrying immortalizing gene)
The position where the immortalizing gene (Bmi-1 gene, TERT gene, and SV40T gene) is integrated is not particularly limited, but when each immortalizing gene is inserted into multiple separate vectors, the Bmi-1 gene is the NP gene. Upstream, the TERT gene is preferably inserted between the P and M genes, and the SV40T gene is preferably inserted upstream of the NP gene. Also, two or more genes (Bmi-1 and TERT, TERT and SV40T, or Bmi-1 and TERT and SV40T) may be inserted into a single vector.

Further, the SeV vector carrying the immortalization gene may be made into a kit, and the kit can include, for example, a medium or container for cell culture, an instruction sheet describing how to use the kit, and the like.

(Introduction of immortalization gene into cells)
The SeV vector carrying the immortalization gene obtained as described above is introduced into the cells by adding the vector (Sendai virus particles) to the medium of the somatic cells and infecting the cells with the virus. .. Since the dose of the vector varies depending on the cell type, cell density, and amount of medium, the MOI whose infection efficiency is close to 100% may be investigated and determined in advance for each cell to be used.

Alternatively, when the SeV vector is in the form of RNP, it can be introduced into cells by a method such as an electroporation method, a lipofection method, or a microinjection method.

(Culture of immortalized gene-introduced cells)
In the present invention, the method for culturing cells into which an immortalizing gene has been introduced can be carried out according to the method and conditions for culturing normal mammalian somatic cells. The medium used for culturing is not particularly limited, and a medium generally used for maintenance culture or expansion culture of cells and suitable for virus infection may be used, and either a commercially available medium or a self-made medium may be used. good. For example, a basal medium containing components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), specifically Dulbecco's Modified Eagle's Medium (D-MEM) medium. , Dulbecco's Modified Eagle's Medium: Natural Mixture F-12 (D-MEM / F-12) Medium, Glassgow MEM (G-MEM) Medium, Basal Medium Eagle (BME) Medium, Minimum Essential Medium (MEM) Medium, Eagle's minimal essential Examples include medium (EMEM) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, RPMI 1640 medium, Medium 199 medium, αMEM medium, ham medium, Fischer medium, and a mixed medium thereof. In addition, the medium may contain growth factors (FGF, EGF, etc.), interleukins, insulin, transferase, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, antibiotics, if necessary. It may contain a substance (penicillin, streptomycin, etc.) and the like. Further, the medium may be a serum-containing medium or a serum-free medium. From the viewpoint of preventing contamination of heterologous animal-derived components, it is preferable to use serum that does not contain serum or that is derived from the same animal as the cells to be cultured. Alternatively, a serum substitute such as albumin may be used.

The culture method is not limited, but is limited to three-dimensional culture under non-adhesive conditions, for example, suspension culture (for example, dispersion culture, aggregate suspension culture, etc.), or two-dimensional culture under adhesive conditions, for example, flat plate. Examples thereof include culture and culture in which three-dimensional culture and two-dimensional culture are combined. The incubator used for culturing cells is not particularly limited as long as it can cultivate cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. .. The incubator may be cell non-adhesive or adhesive, and is appropriately selected depending on the intended purpose. As the cell adhesion incubator, one treated with a cell-supporting substrate or the like using an extracellular matrix or the like may be used for the purpose of improving the adhesion to cells. Examples of the cell-supporting substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.

The culture temperature is 30 ° C to 36 ° C, preferably 32 ° C to 35 ° C, and more preferably 33 to 35 ° C. Culturing is carried out in an atmosphere of CO ₂ containing air, for example, at a CO ₂ concentration of 2% to 5%. The culture temperature for removing the immortalizing gene is 37 ° C to 38 ° C, preferably 37 ° C to 37.5 ° C.

2. 2. Induction of Differentiation According to the present invention, the immortalized cells prepared as described above can be induced to differentiate into specific tissue cells by culturing them in a differentiation-inducing medium. For example, when the immortalized cell is a mesenchymal stem cell, it can be differentiated into adipocyte, osteoblast, nerve cell, chondrocyte and the like.

The composition of the medium for inducing the differentiation of the immortalized cells according to the present invention into the target cells, the differentiation-inducing factor, the culture method, the passage method, etc. can be appropriately set from well-known and conventional techniques.

The differentiation-inducing medium can be appropriately selected according to the type of cells targeted for differentiation-inducing. Differentiation-inducing media for various tissues (mediums to which at least one type of differentiation-inducing or promoting factor according to the target cell for differentiation-inducing is added) are commercially available, and these commercially available media may be used. For example, the medium for inducing the differentiation of immortalized cells according to the present invention into adipocytes is a commercially available adipocyte-inducing medium or a commercially available animal cell medium containing insulin, dexamesazone, indomethacin, or 3-isobutyl-1-methylxanthin. , A medium containing troglycazone, biotin, etc. can be used. Examples of commercially available media include Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (manufactured by PromoCell), Human Mesenchymal Stem Cell Adipogenic Differentiation Medium BulletKit8 (manufactured by Lonza), and the like. The medium for inducing the differentiation of the cells according to the present invention into osteoblasts includes dexamesazone, ascorbic acid, β-glycerophosphate, hydrocortisone, BMP4, in a commercially available osteoblast-inducing medium or a commercially available animal cell medium. A medium containing BMP2 etc. can be used. Examples of commercially available media include Mesenchymal Stem Cell Osteogenic Differentiation Medium (manufactured by PromoCell), Human Mesenchymal Stem Cell Osteogenic Differentiation Medium Bullet Kit (manufactured by Lonza), and the like. As a medium for inducing differentiation of immortalized cells into nerve cells according to the present invention, a commercially available nerve cell culture medium or nerve differentiation medium (for example, Mesenchymal Stem Cell Neurogenic Differentiation Medium (manufactured by PromoCell), etc.) can be used. Further, the nerve cell culture medium or the nerve differentiation inducing medium preferably contains a nerve cell inducing factor (for example, brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF)). Further, as the medium for inducing the differentiation of the immortalized cells according to the present invention into chondrocytes, a commercially available chondrocyte-inducing medium or a commercially available animal cell medium containing dexamesazone, ascorbic acid, and TGF-β3 is used. can. Examples of commercially available media include MesenchymalStemCellChondrogenic DifferentiationMedium (manufactured by PromoCell), HumanMesenchymalStemCellChondrogenicDifferentiationMedium BulletKit (manufactured by Lonza), and the like.

The culture conditions for inducing differentiation are the same as the culture conditions for culturing normal stem cells. The culture period for inducing differentiation is also not particularly limited, but is generally 5 to 20 days, preferably 7 to 18 days.

Whether or not the stem cells have been induced to differentiate into the target cells can be confirmed by examining the expression of markers specific to each differentiated cell. For example, differentiation into adipocytes can be confirmed by oil red 0 staining, differentiation into osteoblasts by alkaline phosphatase staining, differentiation into nerve cells by NeuroFluorNeuO staining, and differentiation into chondrocytes by alcian blue staining.

The immortalized cells and tissue cells induced to differentiate from immortalized (stem) cells obtained by the present invention can be used, for example, by transplanting cells to a diseased or damaged site, and can be provided as a product for regenerative medicine. Examples of products for regenerative medicine include cultured skin, cultured cartilage, cultured corneal epithelium, various cell sheets (for example, epidermal cell sheet, fibroblast sheet, corneal endothelial cell sheet, myocardial cell sheet, osteoblast sheet, myoblast). Cell sheet, nerve cell sheet, cartilage cell sheet, hepatocyte sheet, pancreatic island cell sheet, root membrane cell sheet, etc.) and the like.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.
In the following examples, the serum-containing medium for culturing human mesenchymal stem cells (MSC) is D-MEM (Low Gulucose), 20% FBS, 0.01 mol / L Hepes, Penicillin 100units / ml, Streptomycin 100 μg / It was prepared with a composition of ml and bFGF 20 ng / ml. Since bFGF has a very short half-life, Gibco Heat Stable Recombinant Human bFGF (Thermo Fisher Scientific), which has excellent stability, was used. MSCs were cultured in the above medium in a 5% CO ₂ incubator. The human MSC used in the experiment is either bone marrow-derived, adipose tissue-derived, cord blood-derived, or umbilical cord matrix-derived.

(Example 1) Preparation of seV vector carrying an immortalizing gene and examination of infection conditions (1) Preparation of a SeV vector carrying an immortalizing gene Bmi-1 (B lymphoma Mo-MLV insertion region 1 homolog), which is widely used as an immortalizing factor. ), HTERT (human telomerase reverse transcriptase), SV40T (simian virus 40 large T antigen), E6 / E7 (human papillomavirus 16 E6 protein and E7 protein) were selected, and Bmi was used as the SeV vector for the non-chromosomal RNA virus vector. -1 gene (SEQ ID NO: 1), hTERT gene (SEQ ID NO: 2), SV40T gene (SEQ ID NO: 3), and E6 / E7 gene (SEQ ID NO: 4) were integrated. ID Pharma Co., Ltd. (Headquarters: Tsukuba City) was commissioned to mount the immortalizing gene on the SeV vector and manufacture the vector. In addition, for 3 types of vectors out of 4 types of vectors, the fluorescent dye genes ORF (red), GFP (green), and BFP (blue) are also loaded at the same time, and the presence or absence of gene expression (vector genome) can be easily checked with a microscope. I made it possible to confirm. FIG. 1 shows the structure of each vector. Regarding the position of the immortalizing gene loaded on the SeV vector, high expression results in long-term expression, and in the SeV vector, loading upstream of the vector genome results in higher expression. Therefore, Bmi-1, SV40T is the vector genome. It was installed at the most upstream of. The mounting positions of hTERT and E6 / E7 were changed downstream due to the decrease in vector production efficiency, which is thought to be due to high expression.

For the SeV vector, we used a TS15ΔF type vector that was improved so that the viral vector disappeared from the cells by changing the culture temperature from 35 ° C to 37 ° C (Efficient generation of transgene-free human induced pluripotent stem cells). iPSCs) by temperature-sensitive Sendai virus vectors. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, Takada N, Inoue M, Hasegawa M, Kawamata S, Nishikawa . 2011 Aug 23; 108 (34): 14234-9.).

(2) Examination of SeV vector infection conditions When transfecting with SeV vector, the infection efficiency changes greatly depending on the cell type. In addition, over-infection may cause damage depending on the cell type. Therefore, in order to determine the infected MOI (Multiplicity of Infection: number of vector particles per cell) in advance for the cell type to be used, the infected MOI in MSC was first examined.

Commercially available bone marrow-derived mesenchymal stem cells (product name: ultra-high-purity human mesenchymal stem cells (REC), PuREC Co., Ltd.) are placed in a 48-well plate (FALCON 353230) supplemented with a medium of 200 μl / well in 5 × 10 ⁴ Sprinkle individual / well (70-80% confluent) and add 4 types of immortalizing factor gene-laden SeV vectors to the cells so that the MOI of each vector becomes 1,5,20 by single or multiple co-infection. , Incubated overnight. The next day, the medium was replaced with fresh medium, and the medium was replaced every 2 days thereafter. The medium was expanded to a 12-well plate (FALCON 353043) on the 5th day after the vector infection, and the cells were observed 9 days after the infection.

The results are shown in Figures 2-4. Nine days after infection with the SeV vector, no cytotoxicity was observed in all wells, and no abnormality was observed at MOI: 20 (MOI: 80 for the SeV vector itself). When the infection efficiency of the SeV vector was observed with an inverted fluorescence microscope (Nikon) by the expression of the fluorescent dye, the expression of the fluorescent dye was observed in most cells at MOI: 20 in the single vector infection (Fig. 2). Similar results were obtained with the two-factor co-infection of Bmi-1 and hTERT (Fig. 3). Similar results were obtained with the three-factor co-infection of Bmi-1, hTERT and SV40T (Fig. 4). From this, it was considered that gene transfer into almost 100% of cells was possible when infected with MOI: 20. Therefore, the MOI for subsequent SeV vector infection to MSCs was determined to be 20.

In addition, among the combinations of SeV vectors examined, for co-infection of the three vectors Bmi-1, hTERT, and SV40T, at a high infection rate of MOI: 20, cells were more than other combinations including uninfected controls. It was confirmed that the number clearly increased (Fig. 4). The actively dividing cells with MOI: 20 were smaller in size than the cells without MOI: 1 fluorescence, and were positive for ORF and GFP fluorescence at the same time. In other words, it was speculated that this promotion of cell proliferation was due to the action of three immortalizing factors, Bmi-1, hTERT and SV40T.

(Example 2) Selection of immortalizing factor MSC by infecting MSC with a SeV vector carrying four kinds of immortalizing factor (Bmi-1, hTERT, SV40T, E6 / E7) genes alone and culturing for a long period of time. The factors necessary for immortalization of the disease were selected.

MSC derived from umbilical cord blood (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)) was used for the study, and one or a combination of two or more SeV vectors carrying the immortalizing factor gene was used. (15 ways in total) were infected with the above MSC (48 well plate, 3 × 10 ⁴ / well, MOI: 20). MSCs not infected with SeV vector were used as negative controls. With the proliferation of MSC, the cells are expanded and cultured in the order of 48 well → 12 well → 6 well, and after that, when the cells become confluent (the adhesive surface of the plate is 100% of the cells), the number of cells is measured and subcultured to obtain a total of 16 types of cells. At the same time as measuring the growth rate of the cells, the morphology of the cells was observed under a microscope.

The cell proliferation rate was measured over 80 days from the introduction of the immortalizing gene (number of cells at the time of introduction: 3 × 10 ⁴ cells). The results (No. 1 to No. 16) arranged in descending order of the number of cells at 80 days are shown in Table 1 below together with the number of cells.

The cell condition was observed at about 2 months (59th day) when the cell proliferation of the negative control stopped, and the uniformity of cell size and the degree of dead cells detached from the culture plate were observed in two stages. Judgment was made (Fig. 5). The judgment results are shown in Table 1 (cell size is uniform and cell peeling is small: 〇, cell size is non-uniform and cell peeling is large: ×, presence or absence of immortalizing factor is present. Notated by + or-).

As shown in Table 1 and FIG. 5, all cell morphological abnormalities were observed in combinations without hTERT factor (including negative control), and hTERT is considered to be essential for cell immortalization using SeV vector. rice field. In addition, as a result of comparing the combination of the cell number and the immortalizing factor, the cell proliferation effect of E6 / E7 was hardly observed among the four immortalizing factors.

From the above results, combinations of immortalizing factors suitable for immortalization using the SeV vector were extracted from Table 1 and summarized in Table 2 (No. 16 is shown for comparison as a negative control).

FIG. 6 shows the cell growth curve. In the negative control (No. 16), the growth stopped after about 2 months (66 days), but the growth of the immortalizing factor-introduced cells (No. 1, 2, 4, 12) was prolonged. Although the growth of hTERT gene-introduced cells (No. 12) was prolonged, the growth rate gradually decreased after 2 months, and further growth could not be expected. On the other hand, the combination-introduced cells (No. 1, 2, 4) of Bmi-1 and hTERT, hTERT and SV40T, and Bmi-1 and hTERT and SV40T proliferated stably without slowing down even on the 80th day. Was observed. Among them, the growth rate of the combination of the three factors Bmi-1, hTERT and SV40T was the fastest.

Using human umbilical cord matrix-derived mesenchymal stem cells (product name: HumanMesenchymal Stem Cells from Umbilical Cord Matrix (hMSC-UC), Promo Cell, product code C-12971) instead of the above cord blood-derived MSC. When the combination of immortalizing factors was examined, the proliferation of cells immortalized by the combination of Bmi-1, hTERT and SV40T was also the best, and the cells were uniform and in the best condition by microscopic observation.

(Example 3) Analysis of immortalized cultured cells using a SeV vector carrying a 3-factor (Bmi-1 / hTERT / SV40T) gene (1) Preparation of 3-factor (Bmi-1 / hTERT / SV40T) -introduced immortalized cells In the examination of MSCs derived from different tissues, it was judged that the SeV vector carrying the three-factor (Bmi-1 / hTERT / SV40T) gene was the most suitable for immortalization, and detailed property analysis of the MSCs immortalized with this vector was performed. .. The cells used were MSCs derived from cord blood (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)) used in the selection of immortalizing factors, but the cells were longer than those used in the selection. Cultured (less than 1 month) cells were used. A 3-factor (Bmi-1 / hTERT / SV40T) gene-loaded SeV vector was introduced under the same MOI: 20 conditions as in Example 2. MSCs without gene transfer with SeV vector were simultaneously cultured as a negative control.

(2) Comparison of growth rate by change in culture temperature The immortalized cell line introduced with 3 factors (Bmi-1 / hTERT / SV40T) was cultured in a CO ₂ incubator at 35 ° C using a 6 cm dish, and the plates adhered. When the surface was grown to about 80% of the cells, 1/5 was subcultured to a new 6 cm dish, and the culture was continued for 75 days.

The SeV vector carrying the immortalization gene is a temperature-sensitive vector, and it is possible to rapidly eliminate the intracellular SeV vector genome by raising the culture temperature from 35 ° C to 37 ° C (Efficient generation of transgene-free). human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, Takada N, Inoue M, Hasegawa Natl Acad Sci U S A. 2011 Aug 23; 108 (34): 14234-9). Therefore, in order to confirm whether the SeV vector genome can be removed even in MSC, temperature changes were performed in the culture of immortalized cells introduced with the above three factors (Bmi-1 / hTERT / SV40T), and changes in the number of cells were investigated. rice field.

To examine the removal of the SeV vector due to temperature changes, immortalized cells (day 75) at the time when the proliferative capacity of untreated MSCs decreased were used. During cell passage on the 78th day, two petri dishes seeded with the same number of cells were prepared, one was maintained at 35 ° C and the other was raised to 37 ° C, and the growth patterns of both were raised. Was compared. The results are shown in FIG. In the cells cultured at 37 ° C, the SeV vector was removed (fluorescence disappeared) and the proliferation decreased sharply, but the cells cultured at 35 ° C did not change in particular and proliferated vigorously even after 130 days after gene transfer. (Fig. 7).

(3) Comparison of fluorescent protein expression and cell morphology due to changes in culture temperature When the expression of fluorescent protein was confirmed with a fluorescence microscope 10 days after the temperature change, OFP (simultaneously loaded with Bmi-1) and GFP (simultaneously loaded with hTERT). Fluorescence was confirmed in cells whose culture temperature was maintained at 35 ° C, but its expression was hardly confirmed in cells raised to 37 ° C (Fig. 8).

When the morphology of the cells was confirmed 2 weeks after the subculture (92 days after the start of the culture), the cell status was not changed at 35 ° C and the cells were actively divided, whereas at 37 ° C, the cells were actively divided. A significant difference was observed from the uniformity at 35 ° C. with a noticeable decrease in cell division rate and variation in cell size (Fig. 9).

From the above results, the temperature-sensitive effect of the SeV vector, which has already been clarified in iPS cells, was confirmed by MSC by changing the temperature from 35 ° C to 37 ° C, and at the same time, the cells were immortalized by removing the three immortalizing factors. Was also reset and returned to finitely proliferating cells.

(4) Chromosome analysis Chromosome analysis of cells was performed by the quinacrine-Hoechst fractionation method at the point ▽ in FIG. In the quinacrine-hexist fractionation method, the chromosome slide is first immersed in 50 ml of Macylben solution (280 ml of 0.1 M citrate solution and 220 ml of 0.2 M disodium hydrogen phosphate solution are mixed and autoclaved), and then 50 ml of Macylben. It was soaked in a solution of Hoechst 33258 (cat: B-2883-25MG, Sigma) at 10 ng / ml for 30 minutes. After washing with tap water with a weak water flow from the back of the chromosome slide, soak it in the Makiruben solution again for 5 minutes, and then cover it with a Makiruben encapsulant (mixing Makiruben and glycerol 1: 1) with a cover glass. bottom. As a result of analysis using a chromosome analysis microscope (model: AxioImager Z2, ZEISS) and chromosome analysis software (type: Ikaros V5.7.4 CM / V5.4.12, Metasystems), the immortalized cell line was the parent strain at all points. It was a normal karyotype as well (Fig. 10).

(5) Evaluation of telomere length The telomere length was evaluated at the points marked with * in FIGS. 6 and 7. The telomere length was evaluated by performing real-time PCR using genomic DNA as a template and by relative quantification of telomere sequences. Genomic DNA extraction from cells was performed using Gentra® Puregene® Kit (Qiagen) with reference to the manufacturer's procedure. For real-time PCR, the Telomere primer set and Single copy reference primer set attached to the Relative Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories) were used as primers, and FastStart Essential DNA Green Master (Roche) was used as the PCR reagent. The PCR reaction conditions were as recommended by the manufacturer. StepOnePlus (Life Technologies Japan) was used for PCR reaction and data acquisition, and data analysis was performed with StepOne Software v2.3.

The results are shown in FIG. Telomerase elongation was confirmed with the combination of the three factors Bmi-1, hTERT and SV40T, and with the combination of the two factors Bmi-1 and hTERT, but no elongation was observed with hTERT alone (Fig. 11A). In addition, long-term culture after removal of the immortalizing factor showed a marked reduction in telomeres (Fig. 11B). The final sample ring was similar to the telomere length of the initial hMSC, although shortening was observed even when the immortalizing factor was not removed (35 ° C). From the above results, it was considered that hTERT is necessary for immortalization in comparison with the proliferative ability, but it is not a sufficient condition.

(Example 4) Differentiation ability of immortalized population cells Regarding the immortalized cells (MSC) introduced with the three factors (Bmi-1 / hTERT / SV40T) prepared in Example 3, fat cells, osteoblasts, nerve cells, and chondrocytes. The ability to differentiate into cells was investigated. For the differentiation test, MSC (ATCC PCS-500-010) derived from cord blood that was immortalized with three factors of Bmi-1, hTERT, and SV40T and cultured for a long period of time (cultured for 4 months after gene transfer) was used. Four months is a difficult period for culturing in general MSCs that are not immortalized.

A commercially available (PromoCell) differentiation kit was used for MSC differentiation. Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (product code C-28016) for adipose cell differentiation, Mesenchymal Stem Cell Osteogenic Differentiation Medium (product code C-28013) for osteoblast differentiation, and Mesenchymal Stem Cell Neurogenic Differentiation for nerve cell differentiation. Medium (product code C-28015) and Mesenchymal Stem Cell Chondrogenic Differentiation Medium (product code C-28012) were used for cartilage cell differentiation, and the operation method was carried out according to the attached protocol.

The confirmation of adipocyte differentiation was performed using the fluorescent dye Lipi-Green (Doujin Kagaku: product code LD02), which specifically stains lipid droplets, which is an index of adipocyte differentiation. Osteoblast differentiation was confirmed by alkaline phosphatase staining. Nerve cell differentiation was confirmed using the fluorescent dye NeuroFluor NeuO (VERITAS: product code ST-01801), which specifically stains nerve cells. Chondrocyte differentiation was confirmed by alcian blue staining.

In the early stage cells cultured for about 2 weeks after purchase, MSCs that retain the differentiation potential and have not been gene-introduced in the logarithmic growth phase were used as positive controls, and compared with the differentiation potential of immortalized MSCs that had passed 4 months, fat. In cell differentiation, lipid droplets, which are indicators of differentiation, were observed in both cases. The immortalized MSC had a lower degree of differentiation than the positive control (Fig. 12). In osteoblast differentiation, staining with alkaline phosphatase was confirmed in both immortalized MSC and positive control, and the cells were differentiated into osteoblasts (Fig. 13). Immortalized MSCs had a higher differentiation rate than positive controls. In neuronal differentiation, fluorescence was observed in almost all cells in both immortalized MSC and positive control, indicating that they differentiated into neurons (Fig. 14). Regarding chondrocyte differentiation, although the degree of differentiation between immortalized MSC and positive control cannot be compared, the differentiated MSC clearly stains deeply with alcian blue and becomes chondrocyte when compared with no negative control without induction of differentiation. Differentiation was confirmed (Fig. 15). Based on the above results, the three-factor (Bmi-1 / hTERT / SV40T) -introduced immortalized cells (MSCs) maintained pluripotency even in MSCs that had been immortalized for 4 months, as in the positive control MSCs. It was confirmed that there was.

(Example 5) Cloning test of immortalized MSCs For the three-factor (Bmi-1 / TERT / SV40T) -introduced immortalized cells (MSCs) prepared in Example 3, whether cloning is possible or whether the MSCs are not immortalized We conducted a confirmation experiment to see how difficult cloning is.

MSC (ATCCPCS-500-010) derived from umbilical cord blood that retains differentiation potential and has not been gene-introduced in the logarithmic growth phase in early cells cultured for about 2 weeks after purchase, has just been gene-introduced ( Cloning was performed using three types of MSCs: an immortalized MSC (2 weeks old) and an immortalized MSC (4 months old) cultured for a long time after gene transfer.

The three types of MSCs that had been cultured and maintained were once completely separated into single cells, and the number of cells was measured. The cells were seeded at a variable number such as 32 cells / 100 μl, and cultured in a 5% CO ₂ incubator at 35 ° C until single cells formed colonies. For medium exchange, half the amount was exchanged every 3 days so as not to disturb colonization.

When cultured for 10 days and observed under a microscope, the single cell grew to a colony of sufficient size. Therefore, the number of colonies formed in each well was measured, and the state was schematically summarized in the figure (FIGS. 16 and 17). The numbers in 96 wells in the figure indicate the number of colonies formed.

Looking at this result, it is possible to judge the ease of colony formation at a glance. Non-immortalized early growth MSCs corresponded to common MSCs, but only 2 wells of 32 cells / well compartments were formed. On the other hand, in MSCs into which the immortalizing gene was introduced with the SeV vector, colonies were formed at a high frequency of 9 well / 24 well even at a low density of 4 cells / well in the two weeks immediately after the introduction (Fig. 16). After 4 months, colonies were formed in 16 wells / 24 wells and more than half of the wells even at a low density of 4 cells / well (Fig. 17).

From the above results, it was clarified that even in MSCs that are difficult to clone, if three types of immortalizing genes are introduced with the SeV vector, cloning can be easily performed regardless of the elapsed time thereafter. The ability to clone at any time is very useful from the viewpoint of quality control in regenerative medicine.

(Example 6) Various analyzes of clone immortalized MSC (1) Proliferative ability of cloned cells Five clones of MSC cloned as a single cell 2 weeks after infection with SeV vector were divided into 35 ° C and 37 ° C after culturing for 60 days. Was cultured. Three of the five clones proliferated and decreased with the removal of the SeV vector at 37 ° C. A growth curve is shown for clone A10 among them (Fig. 18).

(2) Confirmation of removal of SeV vector due to temperature change At 35 ° C, the presence of SeV vector could be confirmed by fluorescence observation as in the group culture experiment, but the presence of SeV vector could not be confirmed in the group at 37 ° C.

(3) Chromosome analysis of cloned cells As a result of chromosomal analysis of 10 cloned clones, 8 clones were normal karyotype. Among them, the results of karyotype analysis are shown for clone A10 showing cell proliferation shown in FIG. 18 (FIG. 19).

(4) Differentiation ability of cloned immortalized MSCs Among the cloned immortalized MSCs, 5 clones were examined for their ability to differentiate into fat cells, osteoblasts, and nerve cells by the same method as in Example 4. As a result, the degree of differentiation was also different, just as the morphology was different depending on the clone. However, in all the clones, differentiation into adipocytes, osteoblasts, and nerve cells was observed, and pluripotency was maintained (Fig. 20). Some clones are much better differentiated than the cells before cloning, and in neural differentiation, not only the expression of neural markers but also the appearance of neurites forming a network of neural networks. Observed.

(Example 7) Immortalization and single-cell cloning of MSCs with different cell ages (aging degree) Since the cell populations grown from one MSC have the same genetic properties, the quality is constant and quality control becomes easy. In that respect, single-cell cloning has important significance in regenerative medicine, and single-cell cloning is indispensable in that when a gene is introduced from the outside, only the target cell is selected.

MSC isolated from the human body is a mixture of cells of various stages, from young cells to aged cells. The ratio also varies from individual to individual, and it is difficult to extract and proliferate only cells at a specific time (youth). Conversely, if MSCs can be immortalized at any time from young cells to senescent cells and can be further cloned, their application range will be greatly expanded in regenerative medicine.

Therefore, we immortalized three types of cells, from young cells to aged cells, or in the middle of the period, and investigated whether single-cell cloning is possible.

The cells used were MSCs derived from umbilical cord blood (product name: Umbilical Cord-Derived Mesenchymal Stem Cells; Normal, Human (ATCC PCS-500-010)). The purchased cells were cultured for a long period of about 80 days at 37 ° C until proliferation stopped. During that time, a part of the cells was cryopreserved at about every other week of passage (Fig. 21).

From the cryopreserved cells, three types of cells, early, middle and late (Day 9,24,49), were thawed at the same time, cultured for 11 days to suppress the effects of freezing and thawing, and seeded on a 24-well plate ( After 24 wells (1 × 10 ⁵ cells / well), the next day, the SeV vector carrying the 3 factor (Bmi-1 / hTERT / SV40T) gene was infected with MOI: 20 and immortalized (Day 21, 36, 61).

After inducing immortality, the cells were transferred to a CO ₂ incubator at 37 ° C to 35 ° C and expanded and cultured for 8 days (24 well → 6 well). After that, the immortalized cells were seeded on a 96-well plate so that the number of cells per well was 1, 2, or 4, and the number of non-immortalized control cells was 5, 10 per well. The cells were seeded to 15 or 20 cells, cultured for about 2 weeks until one cell divided and proliferated and a cell population (colony) appeared, and the number of colonies generated by microscopic observation was counted.

The morphology of SeV vector-infected cells and non-SeV vector-infected cells of MSCs of three different cell ages (early, metaphase, and late) was observed immediately before 96-well plate seeding (8 days after SeV vector infection). The difference was noticeable (Fig. 22).

As shown in FIG. 22, it can be seen that the younger the cells, the higher the cell density reflecting the number of cells, and the proliferation of cells is active. The difference in the number of cells depending on the presence or absence of SeV vector infection did not appear significantly, but it was clearly observed that the number of cells increased in the cells infected with the SeV vector in the late stage cells. In particular, changes in cell morphology and size are remarkable, and in the control of SeV vector non-infection, the cells become large and the division rate decreases with the passage of time, but in SeV vector-infected cells, division is active. The size of the cells became about the same, and it became impossible to distinguish between the early stage, the middle stage, and the late stage by the size.

As a result of cloning by 96 well, it was confirmed that in SeV vector non-infected cells, colonies did not appear in 5 cells per 1 well, and cloning was as difficult as in Example 5. In SeV vector-infected cells, many colonies were also observed on single-cell cloning plates seeded at a rate of 1 per well (FIGS. 23-1, 23-2).

In the early cells, colonies were observed at 30 wells per 96 wells, 47 wells in the middle stage, and 27 wells in the late stage. From this result, it was clarified that the MSC induced to be immortalized by the SeV vector can easily obtain a cell population derived from one cell regardless of the degree of aging.

(Example 8) Confirmation of effect on MSCs in which proliferation has stopped The effect of the SeV vector carrying an immortalizing gene on MSCs in which cell division has completely stopped due to aging was confirmed.

Cells in a cryopreserved cell proliferation state (Day72) were freeze-thawed and seeded on a 24-well plate to suppress the effects of freeze-thaw, and the SeV vector was applied to cells confirmed to not grow cells (Day90). Infected (24 well, 1 x 10 ⁵ / well, MOI: 20). After that, the changes in the cells were observed under a microscope.

In SeV vector non-infected cells (control cells), there was no major change, and it was observed that all cells became round and died. On the other hand, in SeV vector-infected cells, no change was observed for several days, but after one week, many cells changed roundly and seemed to die as they were, but small adhesive cells were observed. After 2 weeks, the cells changed to a vigorously proliferating cell population similar to immortalized cells (Fig. 24).

The cells that started reproliferation were single-cell cloned in the same manner as in Example 7. As a result, although the ratio was lower than the results of FIGS. 23-1 and 23-2 of the above-mentioned single cell, a proliferated cell population was confirmed in 17 wells out of 96 wells, and the cells that completely stopped dividing were also infected with the SeV vector. It was confirmed that the division started again and single cell cloning became possible (Fig. 25).

This change was considered to remove the cytoplasm unnecessary for cell proliferation, unlike cell proliferation associated with cell immortalization. The detailed mechanism is unknown, but since cells that have completely stopped growing due to aging are repopulated, in this case, the expression "cell rejuvenation" rather than "immortalization" is consistent.

(Example 9) Examination of cell proliferation of human mesenchymal stem cells in serum-free medium Adipose tissue-derived human mesenchymal stem cells hMSC-AT (PromoCell; C-12977) and bone marrow-derived human mesenchymal stem cells hMSC-BM (PromoCell) C-12974) was used in the experiment. Assuming that the SeV betaku infection date of hMSC-AT and hMSC-BM is the "infection date", hMSC-AT non-infected cells and infected cells are non-infected cells of hMSC-BM from the infection date to the 13th and 11th days, respectively. Infected cells and infected cells were cultured in a medium containing serum from the day of infection to the 11th and 17th days, respectively. As the serum-containing medium, a medium having a composition of D-MEM (Low Gulucose), 20% FBS, 0.01 mol / L Hepes, Penicillin 100units / ml, Streptomycin 100 μg / ml, and bFGF 20 ng / ml was used. Then, it was replaced with a serum-free medium and the culture was continued. Stem Fit For Mesenchymal Stem Cell (AJINOMOTO; A3) medium was used as the serum-free medium. As the culture dish, a 6 cm dish (CORNING; 353004) coated with iMatrix-511 silk (Matrixome; 892 091) was used.

Infection with the SeV vector was performed on 2 × 10 ⁵ hMSC-AT cells or hMSC-BM cells under the condition of MOI: 40. The non-infection condition and the infection condition were carried out once each. The medium was changed 24 hours after infection and maintenance culture was continued. Cell culture was performed in a 5% CO ₂ incubator at 35 ° C.

In the hMSC-AT cell experiment, the non-infected cell line was found to have a decrease in the number of cells, so the culture was terminated 52 days after the day of infection. On the other hand, the SeV vector-infected cell line continued to proliferate after the 52nd day (Fig. 26).

In the hMSC-BM cell experiment, the cell proliferation of the non-infected cell line stopped and stopped growing after 40 days from the day of infection, and the cell number was measured on the 72nd day. As a result, the number of cells was below the detection limit, and cell culture was terminated at this point. On the other hand, the SeV vector-infected cell line continued to proliferate even on the 40th day (Fig. 27).

From the above results, it is possible to prolong the cell division ability of MSC cells derived from adipose tissue and bone marrow as well as MSC cells derived from umbilical cord blood by SeV vector infection. It was confirmed that the growth could be continued.

After infection with SeV vector, fluorescence observation of GFP (hTERT) and OFP (Bmi-1) was performed. At the initial stage of culturing hMSC-AT cells and hMSC-BM cells in serum-free medium (16th and 24th days from the day of infection) and when culturing in serum-free medium was continued (58th day from the day of infection). When the 50th day) was compared, it was confirmed that the expression of GFP and OFP was maintained (Fig. 28). From this result, it was confirmed that SeV was maintained even if the culture was continued in the serum-free medium as in the case of the culture in the serum-containing medium.

(Example 10) Examination of cell proliferation of immortalized rat MSC Rat subcutaneous fat-derived mesenchymal stem cells rMSC cells (Cosmo Bio Co., Ltd., MSA01C) are media for proliferation of rat subcutaneous fat-derived mesenchymal stem cells (Cosmo Bio Co., Ltd.). Cultivated in MSA-GM).

Infection with the SeV vector was performed on 2 × 10 ⁵ rMSC cells under the condition of MOI: 40. The non-infected condition was once and the infected cell line was twice. The medium was changed 24 hours after infection and maintenance culture was continued. Culture dishes were placed in a collagen coated dish (IWAKI, 4810-010) and maintained at 35 ° C. in a 5% CO ₂ incubator.

When long-term culture was carried out, a phenomenon was observed in which the rMSC cell line, whose proliferative ability originally decreased after several passages, continued to proliferate in non-infected cells (Fig. 29). This is thought to be due to the accidental immortalization of the rMSC cell line. However, as a result of culturing for 75 days, it was found that the infected cells grew more than twice as fast as the non-infected cells.

After infection with SeV vector, fluorescence observation of GFP (hTERT) and OFP (Bmi-1) was performed. Fluorescent photographs were taken with an all-in-one microscope (KEYENCE; BZ-X800). The photographs were taken under the conditions that the magnification was 20 times and the exposure times of GFP and OFP were 1/5 second and 1/20 second, respectively. As a result of fluorescence observation, the expression of OFP (Bmi-1) and GFP (hTERT) was observed under the condition of 35 ° C. even after culturing for 58 days after infection (Fig. 30).

(Example 11) Examination of cell proliferation of immortalized HFL1 Human fibroblasts (HFL1 cells, RIKEN: RCB0521) were deactivated in ham-F12 medium (Nakalai; 17458-65) (NICHIREI; 175012). Was cultured in a medium containing 15% (v / v) of penicillin-streptomycin solution (Fuji Film Wako; 168-23191) at 100 units / mL.

For infection with the SeV vector, 2 × 10 ⁵ HFL1 cells were infected with the SeV vector under the condition of MOI: 40. The non-infection condition and the infection condition were carried out twice each. The medium was changed 24 hours after infection and maintenance culture was continued. Infected cells were divided into two conditions, one was to culture at 35 ° C and the other was to culture at 37 ° C on the 34th day after infection (Fig. 31, arrow). Cell culture was performed at each temperature in a 5% CO ₂ incubator.

Since the number of non-infected cells decreased, the culture was terminated on the 148th day from the day of infection. On the other hand, in the infected cell line, the cells cultured at 37 ° C. stopped proliferating, so that the culture was completed on the 259th or 273th day from the day of infection (FIG. 31). From this, it was clarified that the cell division ability can be prolonged even in HFL1 cells by SeV vector infection.

By calculation, it was investigated how much the cells had grown since the culture was started. As a result, the growth of non-infected cells stopped at the 17th order, whereas the growth of non-infected cells stopped at the 31st order in the SeV vector-infected 37 ° C culture. In the SeV vector-infected 35 ° C culture, the cells grew to around 43 orders without stopping even during the culture period, and a difference of 26 orders occurred in the cell number comparison with the non-SeV vector-infected cells in 260 days. From the above results, it was confirmed that in human fibroblast HFL1, more cells can be proliferated by SeV vector infection.

After infection with SeV vector, fluorescence observation of GFP (hTERT) and OFP (Bmi-1) was performed. The result is shown in FIG. At the 34th day from the day of infection, the fluorescent signals of GFP and OFP similar to those of the cells on the 10th day from the day of infection were shown. The cells were further cultured for 221 days at 35 ° C and 37 ° C, respectively. Cells cultured at 35 ° C had significantly reduced GFP expression compared to cells on days 34 and 255, but OFP was maintained as on days 10 and 34. .. On the other hand, the positive rates of GFP and OFP were significantly reduced in the cells cultured at 37 ° C. as compared with the cells on the 10th and 34th days. From these results, it was confirmed that SeV was removed in a temperature-sensitive manner.

In general, cells with advanced cellular senescence may exhibit characteristics such as an increase in cell size, a flat shape, and the formation of vacuoles. Infected cells cultured at 35 ° C showed no characteristic of cellular senescence compared to infected cells cultured at 37 ° C. From this result, it was confirmed that the cells continued to be cultured at 35 ° C. did not undergo cell senescence in terms of cell morphology.

(Example 12) Examination of cell proliferation of immortalized HUVEC Human umbilical vein endothelial cells (HUVEC cells, Promocell, C-12205) were cultured in an endothelial cell medium (ScienCell; 1001).

Infection with the SeV vector was performed on 2 × 10 ⁵ HUVEC cells under MOI: 40 conditions. The non-infection condition was once and the infection condition was twice. The medium was changed 24 hours after infection and maintenance culture was continued. Infected cells were divided into two conditions, one was to culture at 35 ° C and the other was to culture at 37 ° C on the 35th day after infection (Fig. 33, arrow). Cell culture was performed at each temperature in a 5% CO ₂ incubator.

The non-infected cells were found to have a decrease in the number of cells, so the culture was terminated on the 39th day from the day of infection. On the other hand, the infected cells continued to proliferate even 74 days after the infection (Fig. 33). From this, it was confirmed that cell proliferation can be prolonged even in HUVEC cells by SeV vector infection.

By calculation, it was investigated how much the cells had grown since the culture was started. As a result, the growth of non-infected cells stopped at around 8 orders, whereas the growth was up to 18 orders in SeV vector-infected 37 ° C culture. In the SeV vector-infected 35 ° C culture, the cells continued to proliferate and proliferated to around 21 orders, and in 74 days, there was a difference of 13 orders in the number of cells from the cells not infected with SeV vector. From the above results, it was confirmed that in human umbilical vein endothelial cells HUVEC cells, more cells can be proliferated by SeV vector infection.

After infection with SeV vector, fluorescence observation of GFP (hTERT) and OFP (Bmi-1) was performed. The result is shown in FIG. 34. At the 36th day from the day of infection, the cells continued to be cultured at 35 ° C. showed the same GFP and OFP fluorescence signals as the cells on the 7th day from the day of infection. After that, when the cells were cultured at 35 ° C and 37 ° C for another 37 days, the proportion of cells that continued to be cultured at 35 ° C was fluorescence-positive compared to the cells on the 7th and 36th days from the day of infection. However, a fluorescent signal was observed in many cells. On the other hand, in the cells cultured at 37 ° C, the proportion of fluorescence-positive cells was significantly reduced as compared with the cells on the 7th and 36th days. From this, it was confirmed that SeV was removed in a temperature-sensitive manner.

(Example 13) Introduction of a multi-growth factor-inserted human artificial chromosome vector into immortalized MSC cells, stability after long-term culture, and induction of differentiation

(1) Chromosome transfer by fusion of micronuclear cells and isolation of drug-resistant clones HGF (hgf), GDNF (gdnf), IGF-1 (Hgf), GDNF (gdnf), IGF-1 described in Watanabe at al. (Mol Ther Nucleic Acids. 2015) as chromosome-donating cells. CHO cells carrying 21HAC2 carrying igf-1), and luciferase (e-luc) were used, and hMSC-UC No. 3 cells, which are immortalized MSC cells described in Example 1, were used as chromosome receptor cells. , Katoh et al. (BMC Biotechnology, 2010, 10:37). Micronuclear cell fusion and culture were performed by the method described. After culturing under BS selective culture for 1 week, resistant colonies appeared, and a total of 9 colonies obtained by 4 fusions were isolated and proliferated, and the subsequent analysis was performed.

(2) Confirmation of transferred chromosomes (2-1) Observation with fluorescence microscope When nine cloned colonies were observed under a fluorescence microscope, GFP-positive cells were observed in all clones, and the positive rate was 50 to 100. %Met.

(2-2) FISH analysis 4 clones (clone names: hMSC-UC No.3 # 1-01, # 1-02, # 2-01, # 3-01) confirmed by PCR analysis are HGF (hgf), FISH analysis was performed using a PAC carrying GDNF (gdnf), IGF-1 (igf-1), and luciferase (e-luc) as a probe. It was confirmed that there were 2 clones (Fig. 35, hMSC-UC No.3, # 3-01).

(3) In vitro differentiation induction The above 4 clones confirmed by PCR analysis were induced to differentiate into bone, cartilage, and adipocytes according to the method of Okamoto et al. (BBRC, 295: 354, 2002), and the differentiation potential equivalent to that of the parent strain was obtained. You can check whether it is maintained or not.

From the above analysis results (1) to (3), it was confirmed that two clones of immortalized MSC carrying a multi-growth factor-inserted human artificial chromosome vector were obtained by micronucleus cell fusion.

(Example 14) Verification of enteritis healing effect of immortalized MSC in an inflammatory bowel disease model (1) CD4 positive CD45RB high positive CD25 negative (CD4 ⁺ CD45RB ^{High +} CD25- ⁾ Isolation of T cells 8 weeks old BALB / cAJcl ( After acclimatization of 30 females of Claire Japan) for 1 week, blood was removed by cardiac blood sampling under anesthesia, and the spleen and mesenteric lymph nodes were collected. The spleen was hemolyzed after tissue dispersion using the MACS system (Miltenyi Biotech). The mesenteric lymph nodes were crushed with a 1 mL syringe plunger and filtered through a 40 μm cell strainer. Antibodies (APC-H7 Rat anti-) to isolate CD4 + CD45RB High + CD25-T cells after combining spleen and mesenteric lymph node cells and separating CD4 positive cells by CD4 microbeads (Miltenyi Biotech) treatment. Labeled with mouse CD4 antibody (BD), FITC Rat Anti-Mouse CD45RB antibody (BD), PE / Cy7 anti-mouse CD25 antibody (BioLegend)) and fractionated by flow cytometer (MoFlo XDP, BECKMAN).

(2) CD4 ⁺ CD45RB High + CD25 --T cell transfer 9-week-old female SCID (CB-17 / lcr-scid / scidJcl: Japan Claire), 4.0 x 10 ⁵ CD4 + CD45RB ^{High +} CD25 ^- T per animal Cells were transplanted by tail vein injection (cell transplant group: 1 group 8 animals x 5 groups, untreated group (PBS administration): 1 group x 7 animals). After transplantation, body weight was measured three times a week and the coat and stool condition were observed. Twenty-one days after cell administration, randomization was performed using the "multivariable block allocation" system of the statistical analysis software JMP based on body weight changes, and the cell transplantation group was divided into groups. At that time, individuals who deviated from the average relative body weight of ± 2 SD were excluded.

(3) Culture of transplanted cells In order to obtain cells for transplantation, hMSC-UC (parent MSC) and immortalized MSC were cultured under each cell culture condition.

(4) MSC administration (therapeutic cell transplantation)
On the 21st, 28th, and 35th days after transplantation of CD4 + CD45RB ^{High +} CD25-T cells, parental MSC and immortalized MSC were administered 1.0 × 10 ⁶ per mouse by tail vein injection. As a positive control, Dexamethasone (Sigma): Dex (1 mg / kg, 100 μL / animal) was subcutaneously administered from the 21st day to the 14th day. Dex solution was prepared at the time of use based on the group average body weight on the 21st and 28th days.

(5) Collection On the 42nd day from the start of the experiment, all blood was collected and serum was prepared (stored at -80 ° C). Furthermore, the gastrointestinal tract between the pyloric ring and the anus (colon to rectum) was sampled, and after evaluation of the condition and measurement of weight and length, the gastrointestinal tract was fixed with 10% formalin.

The results are shown in FIG. As a result of observing the therapeutic effect of MSC transplantation on the IBD model of adoptive immunotransplantation using SCID mice, the clinical score value (clinical score value) in the Dex-administered group, the parent MSC-administered group, and the immortalized MSC-administered group compared with the non-treated group ( Weight loss score, thickening score, stool score, and coat score) decreased significantly. In particular, in the parent MSC-administered group, the decrease was nearly 1/2, and a high effect on improving enteritis was expected. There was no significant difference between the immortalized MSC-administered group and the parent MSC-administered group, which was equivalent to Dex administration. rice field. Regarding the culture of cells for transplantation, immortalized cells proliferated significantly better than the parent cells in the early stage of culture, and it did not take time to prepare the cells.

The present invention can be used in the fields of cell medicine and regenerative medicine. The cell immortalization technique of the present invention can be used not only for normal cells with slow cell proliferation but also for cancer cells and the like, and can also be used in the field of basic research. Furthermore, single-cell cloning, which is essential for gene transfer and chromosome transfer, is also possible. In addition, the cells obtained by the present invention continue to proliferate without aging, which facilitates quality control, promotes mechanization of culture, significantly reduces cell production costs, and handles a large number of cells. Is possible. Therefore, the present invention expands the range of applicable diseases in cell medicine and regenerative medicine, and contributes to the activation of domestic and foreign medical-related industries.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A method for producing reversible immortalized cells, which comprises the following steps.
(1) A step of introducing a chromosome-non-integrating RNA viral vector carrying an immortalizing gene into mammalian cells and expressing the immortalizing gene in the cells, and (2) culturing the cells obtained in step (1). And the process of growing
The method according to claim 1, wherein the immortalizing gene is one or more immortalizing genes selected from the group consisting of the Bmi-1 gene, the TERT gene, and the SV40T gene.
The method according to claim 1 or 2, wherein the immortalizing gene is any one of the following (a) to (d).
(a) Combination of Bmi-1 gene, TERT gene, and SV40T gene
(b) Combination of Bmi-1 gene and TERT gene
(c) Combination of TERT gene and SV40T gene
(d) TERT gene
The method according to any one of claims 1 to 3, wherein the cell is a somatic cell.
The method according to claim 4, wherein the somatic cell is a somatic stem cell.
The method according to claim 5, wherein the somatic stem cell is a mesenchymal stem cell.
The method according to any one of claims 1 to 6, wherein the chromosomal non-integrated RNA viral vector is a negative-strand RNA viral vector.
The method according to claim 7, wherein the negative strand RNA virus vector is a paramyxovirus vector.
The method according to claim 8, wherein the paramyxovirus vector is a Sendai virus vector.
The method according to claim 9, wherein the Sendai virus vector is a temperature-sensitive Sendai virus vector.
The method according to claim 1, wherein the chromosomal non-integrating RNA virus vector is a Sendai virus vector, further comprising a step of removing the Sendai virus vector after culturing in the step (2).
The method according to claim 11, wherein the removal of the Sendai virus vector is performed by changing the culture temperature from 35 ° C to 37 ° C.
The method according to any one of claims 1 to 12, further comprising a step of cloning immortalized cells after culturing in the step (2).
Immortalized cells obtained by the method according to any one of claims 1 to 13.
Immortalized cells containing a Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of Bmi-1 gene, TERT gene, and SV40T gene in a removable state.
A regenerative medicine product containing the immortalized cell according to claim 14 or 15.
A temperature-sensitive Sendai virus vector carrying one or more immortalizing genes selected from the group consisting of Bmi-1 gene, TERT gene, and SV40T gene.
A kit for producing reversible immortalized cells, which comprises the temperature-sensitive Sendai virus vector according to claim 17.